The long-term goals are to define the ionic and molecular mechanisms that underlie the intrinsic pacemaker activity of the sinoatrial (S-A) node, the pacemaker of the heart, and to explain how such activity is modulated by neurotransmitters, hormones and disease. The focus of the present study will be ionic mechanisms. Three inward currents will be investigated: (1) iCl, a time-independent chloride current activated by catecholamines via a cAMP; (2) iNa, a rapidly activating and inactivating sodium current that is blocked by tetrodotoxin; and (3) i(f), a nonselective current that is activated by hyperpolarization. Two hypotheses will be tested. (1) Because of its time independence and greater amplitude at the maximum diastolic potential, iCl is more likely than i(f) to generate intrinsic pacemaker activity and the "positive chronotropic" effect of beta-adrenoceptor agonists (i.e. increased heart rate). (2) By reducing the threshold for firing, iNa plays a role in the positive chronotropic effect; however, because of its voltage dependence, iNa contributes little to intrinsic pacemaker activity. To meet these objectives, three specific aims will be achieved in single, cultured, primary pacemaker cells isolated from the rabbit S-A node: (1) tests of the hypothesis that cultured, primary pacemaker cells are an accurate model of whole S-A node (not dissociated) or right atrial preparations; (2) use of selective inhibitors to quantify how much each current contributes to intrinsic pacemaker activity and the positive chronotropic effects of isoproterenol (ISO), a beta agonist; and (3) use of both "physiological" and conventional voltage clamp techniques to quantify each current's contribution to intrinsic pacemaker activity and the effects of ISO. Beat rates of non-patched cells will be measured with a video motion-detection system. Spontaneous action potentials and their derivatives will be recorded with the "perforated patch" (nystatin) technique. An "action potential clamp" procedure will measure "compensation" current induced by ISO or current inhibitors, and both perforated (iCl and i(f) and conventional (iNa) patch clamp techniques will be used to measure whole-cell currents. ISO dose-response curves will be obtained for each measured parameter. Knowing which current contributes most to pacemaker activity would allow one to stimulate that current and heart rate selectively, rather than receptors that have multiple effectors. This could reduce side effects in the treatment of sinus bradycardia, which occurs in acute myocardial infarction, heart transplants, severe heart failure and the "sick sinus" syndrome, which is a major cause of morbidity in the elderly and accounts for up to 52% of all permanent pacemakers.